Project Summary Human babesiosis is a malaria-like multisystem disease caused primarily by Babesia microti, an emerging apicomplexan parasite that infects and develops within human erythrocytes. The parasite is transmitted to humans by the tick vector Ixodes scapularis and can also be introduced through blood transfusion. Infection can cause flu-like symptoms, and severe infection can be fatal, in particular in the immunosuppressed and the elderly. Current methods for babesiosis diagnosis include microscopy, PCR, IFA and ELISA-based methods that detect antibodies in serum from patients or donors. Each of these methods has major limitations. PCR detection of the parasite DNA is the most sensitive of all current diagnostic methods used. However, PCR only tests for the presence of parasites within a fraction of a sample being tested (typically 1ml out of 500 ml). Thus, at very low parasitemias, a false negative result is possible when the test sample happens to be devoid of infected red blood cells. In sum, current screening criteria reduce the probability of blood contamination but do not completely eliminate the risk of transmission by transfusion to individuals with a weakened immune system. Here we propose to develop a capture ELISA assay that can detect the most highly expressed and immunogenic antigen of the parasite, BmGPI12/BmSA1. Our preliminary studies using short-term in vitro culture as well as controlled mouse infections demonstrated that a capture assay targeting this antigen detects infection before the parasite is detectable by microscopy or PCR, with parasitemia levels lower than 0.067%. Because each infected cell releases thousands of BmGPI12/BmSA1 molecules into the serum, we expect that our antigen detection assay will prove more sensitive than any method detecting the parasite itself, including PCR. Building upon our preliminary data, we propose the following two specific aims towards the development of a test that could be implemented for high-throughput detection of B. microti in blood donations. In Aim 1, we will optimize the assay procedure using a set of well-characterized blood and sera from infected and non-infected laboratory mice. To achieve regulatory standards of reproducibility in a commercial assay, we will develop monoclonal antibodies against BmGPI12/BmSA1 to replace the polyclonal serum used in preliminary experiments. In Aim 2, we will use the optimized assay to screen a collection of 100 human serum samples available at Yale University and L2 Diagnostics. These samples have previously been characterized by B. microti PCR detection and serology. Our experiments will provide proof of feasibility needed for future efforts and collaborations with major blood organizations to use the assay in large scale blood screening. The success of the proposed studies will set the stage for use of this assay to screen the blood supply to prevent transfusion-transmitted babesiosis.